Engineered cord blood megakaryocytes evade killing by allogeneic T-cells for refractory thrombocytopenia.

The current global platelet supply is often insufficient to meet all the transfusion needs of patients, in particular for those with alloimmune thrombocytopenia. To address this issue, we have developed a strategy employing a combination of approaches to achieve more efficient production of functional megakaryocytes (MKs) and platelets collected from cord blood (CB)-derived CD34+ hematopoietic cells. This strategy is based on ex-vivo expansion and differentiation of MKs in the presence of bone marrow niche-mimicking mesenchymal stem cells (MSCs), together with two other key components: (1) To enhance MK polyploidization, we used the potent pharmacological Rho-associated coiled-coil kinase (ROCK) inhibitor, KD045, resulting in liberation of increased numbers of functional platelets both in-vitro and in-vivo; (2) To evade HLA class I T-cell-driven killing of these expanded MKs, we employed CRISPR-Cas9-mediated ß-2 microglobulin (ß2M) gene knockout (KO). We found that coculturing with MSCs and MK-lineage-specific cytokines significantly increased MK expansion. This was further increased by ROCK inhibition, which induced MK polyploidization and platelet production. Additionally, ex-vivo treatment of MKs with KD045 resulted in significantly higher levels of engraftment and donor chimerism in a mouse model of thrombocytopenia. Finally, ß2M KO allowed MKs to evade killing by allogeneic T-cells. Overall, our approaches offer a novel, readily translatable roadmap for producing adult donor-independent platelet products for a variety of clinical indications.

Targeting the αv integrin/TGF-ß axis improves natural killer cell function against glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-ß activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-ß signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-ß axis as a potentially useful therapeutic target in GBM.

Metabolic Reprogramming of GMP Grade Cord Tissue Derived Mesenchymal Stem Cells Enhances Their Suppressive Potential in GVHD.

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.

Remotely controlled nanofluidic implantable platform for tunable drug delivery.

Chronic diseases such as hypertension and rheumatoid arthritis are persistent ailments that require personalized lifelong therapeutic management. However, the difficulty of adherence to strict dosing schedule compromises therapeutic efficacy and safety. Moreover, the conventional one-size-fits-all treatment approach is increasingly challenged due to the intricacies of inter- and intra-individual variabilities. While accelerated technological advances have led to sophisticated implantable drug delivery devices, flexibility in dosage and timing modulation to tailor precise treatment to individual needs remains an elusive goal. Here we describe the development of a subcutaneously implantable remote-controlled nanofluidic device capable of sustained drug release with adjustable dosing and timing. By leveraging a low intensity electric field to modify the concentration driven diffusion across a nanofluidic membrane, the rate of drug administration can be increased, decreased or stopped via Bluetooth remote command. We demonstrate in vitro the release modulation of enalapril and methotrexate, first-line therapeutics for treatment of hypertension and rheumatoid arthritis, respectively. Further, we show reliable remote communication and device biocompatibility via in vivo studies. Unlike a pulsatile release regimen typical of some conventional controlled delivery systems, our implant offers a continuous drug administration that avoids abrupt fluctuations, which could affect response and tolerability. Our system could set the foundation for an on-demand delivery platform technology for long term management of chronic diseases.

Nanofluidic drug-eluting seed for sustained intratumoral immunotherapy in triple negative breast cancer.

Conventional systemic immunotherapy administration often results in insufficient anti-tumor immune response and adverse side effects. Delivering immunotherapeutics intratumorally could maximize tumor exposure, elicit efficient anti-tumor immune response, and minimize toxicity. To fulfill the unmet clinical need for sustained local drug delivery and to avoid repeated intratumoral injections, we developed a nanofluidic-based device for intratumoral drug delivery called the nanofluidic drug-eluting seed (NDES). The NDES is inserted intratumorally using a minimally invasive trocar method similar to brachytherapy seed insertion and offers a clinical advantage of drug elution. Drug diffusion from the NDES is regulated by physical and electrostatic nanoconfinement, thereby resulting in constant and sustained immunotherapeutic delivery without the need for injections or clinician intervention. In this study, the NDES was used to deliver immunotherapeutics intratumorally in the 4 T1 orthotopic murine mammary carcinoma model, which recapitulates triple negative breast cancer. We demonstrated that NDES-mediated intratumoral release of agonist monoclonal antibodies, OX40 and CD40, resulted in potentiation of local and systemic anti-tumor immune response and inhibition of tumor growth compared to control mice. Further, mice treated with NDES-CD40 demonstrated minimal liver damage compared to systemically treated mice. Collectively, our study highlights the NDES as an effective platform for sustained intratumoral immunotherapeutic delivery. The potential clinical impact is tremendous given that the NDES is applicable to a broad spectrum of drugs and solid tumors.

Efficacy of sustained delivery of GC-1 from a Nanofluidic system in a spontaneously obese non-human primate: a case study.

With nearly 40% of U.S. adults obese, and childhood and adolescent rates rising, obesity and associated comorbidities are serious public health concerns with massive societal costs. Often, lifestyle interventions do not offer sufficient weight loss to improve health, requiring surgery and medications as adjunct management strategies. Here, we present a 4-month case study in which the sustained, low-dose, and constant administration of the thyroid receptor ß selective agonist GC-1 (sobetirome) from a novel nanochannel membrane implant was assessed in an obese, pre-diabetic rhesus macaque. Dramatic loss of white adipose tissue in the abdomen from 36 to 18% was observed via magnetic resonance imaging in conjunction with normalized serum insulin and glycemia, with no signs of cardiotoxicity shown. The non-human primate study highlights sustained low-dose delivery of GC-1 from our minimally invasive subcutaneous implant as a valuable approach to induce weight loss and manage obesity and comorbidities, including type 2 diabetes.

A pharmacokinetic study of GC-1 delivery using a nanochannel membrane device.

This study demonstrated a nanochannel membrane device (NMD) for controlled and sustained release of GC-1 in rats, in the context of the treatment of metabolic syndrome. Release profiles were established in vitro both with and without 5% labrasol for over 2 months. In vivo pharmacokinetic evaluation showed effective GC-1 plasma concentrations, which resulted in significant reductions in body weight after just one week of treatment when compared to the NMD releasing vehicle only (PBS). We also provided evidence that rats treated with NMD-GC-1 present sub-active thyroids and clear differences in the morphology of the epithelium and follicles as compared to the controls, while the heart showed changes in weight. Moreover, body temperatures remained stable throughout treatment, and glucose, pancreatic islet size, and liver histology appeared similar between the treated and control groups. Prolonged constant administration of GC-1 from the NMD proved to be a valid strategy to facilitate weight loss.

Integrated multimodal-catheter imaging unveils principal relationships among ventricular electrical activity, anatomy, and function.

Multiple imaging modalities are employed independent of one another while managing complex cardiac arrhythmias. To combine electrical, anatomical, and functional imaging in a single catheter system, we developed a balloon catheter that carried 64 electrodes on its surface and an intracardiac echocardiography (ICE) catheter through a central lumen. The catheter system was inserted, and the balloon was inflated inside the left ventricle (LV) of eight dogs with 6-wk-old infarction, created by occlusion in the left anterior descending coronary artery. Anatomy was constructed by ICE imaging (9 MHz) through the balloon. Single-beat noncontact mapping (NCM) was performed via the multielectrode array to reconstruct unipolar endocardial electrograms during sinus rhythm. Standard contact mapping (CM) of the endocardium was also carried out for reference. Myocardial infarction in anterior LV extending from the middle to apical regions was localized both by ICE and NCM and validated by CM and pathology. The overall difference in the activation times between NCM and CM was 3 +/- 1 ms. Unipolar voltage in infarcted middle anterior LV was smaller than the voltage in normal middle inferior LV both by NCM (11 +/- 4 vs. 16 +/- 3 mV; P = 0.002) and CM (11 +/- 3 vs. 20 +/- 4 mV; P < 0.001). Unipolar voltage was also inversely related to infarct transmurality, both by NCM (r = -0.87; P = 0.005) and CM (r = -0.94; P < 0.001). The infarct area by ICE (7.7 +/- 2.9 cm(2)) was in agreement with CM (bipolar voltage, <1 mV; and area, 7.6 +/- 3.3 cm(2); r = 0.80; P = 0.016). Meanwhile, the voltage threshold that depicted the infarct area by NCM was directly related to the smallest unipolar voltage reconstructed within the infarct (r = 0.96; P < 0.001). In conclusion, combining NCM and ICE imaging in a single catheter system is feasible. The preclinical development of such an integrated system and its evaluation in experimental myocardial infarction demonstrate capabilities for single-beat mapping at multiple sites as well as the online assessment of anatomy and myocardial function.